Goldberg J M, Baldwin R L
Department of Biochemistry, Beckman Center, Stanford University Medical Center, Stanford, California 94305-5307, USA.
Biochemistry. 1998 Feb 24;37(8):2546-55. doi: 10.1021/bi972402y.
The bimolecular association rate constant (kon) and dissociation rate constant (koff) of the complex between fluorescein-labeled S-peptide analogues and folded S-protein are reported. This is the first kinetic study of a protein folding reaction in which most of the starting material is already folded and only a small part (one additional helix) becomes ordered; it provides a folding landscape with a small conformational entropy barrier, and one in which kinetic traps are unlikely. Refolding and unfolding are measured under identical strongly native conditions, and the reaction is found to be two-state at low reactant concentrations. The dissociation constant (Kd) of the complex and the properties of the transition state may be calculated from the rate constants without extrapolation. The folded complex is formed fast (kon = 1.8 x 10(7) M-1 s-1) and is very stable (Kd = 6 pM) at 10 degrees C, 10 mM MOPS, pH 6.7. Charge interactions stabilize the complex by 1.4 kcal mol-1. The charge effect enters in the refolding reaction: increasing the salt concentration reduces kon dramatically and has little effect on koff. Urea and GdmCl destabilize the complex by decreasing kon and increasing koff. The slopes (m-values) of plots of ln Kd vs [cosolvent] are 0.75 +/- 0.04 and 2.8 +/- 0.3 kcal mol-1 M-1 for urea and GdmCl, respectively. The ratio mon/(mon + moff) is 0.54 +/- 0.04 for urea and 0.57 +/- 0.1 for GdmCl, where mon is the m-value for kon and moff is the m-value for koff, indicating that more than half of the sites for interaction with either cosolvent are buried in the ensemble of structures present at the transition state.
报道了荧光素标记的S肽类似物与折叠的S蛋白之间复合物的双分子缔合速率常数（kon）和解离速率常数（koff）。这是对蛋白质折叠反应的首次动力学研究，其中大部分起始材料已经折叠，只有一小部分（一个额外的螺旋）变得有序；它提供了一个具有小构象熵垒的折叠景观，并且是一个不太可能存在动力学陷阱的景观。在相同的强天然条件下测量重折叠和去折叠，发现在低反应物浓度下反应是两态的。复合物的解离常数（Kd）和过渡态的性质可以从速率常数计算得出，无需外推。折叠的复合物形成迅速（kon = 1.8×10⁷ M⁻¹ s⁻¹），并且在10℃、10 mM MOPS、pH 6.7时非常稳定（Kd = 6 pM）。电荷相互作用使复合物稳定1.4 kcal mol⁻¹。电荷效应参与重折叠反应：增加盐浓度会显著降低kon，而对koff影响很小。尿素和GdmCl通过降低kon和增加koff使复合物不稳定。ln Kd对[共溶剂]作图的斜率（m值），尿素为0.75±0.04 kcal mol⁻¹ M⁻¹，GdmCl为2.8±0.3 kcal mol⁻¹ M⁻¹。对于尿素，kon的m值（mon）与koff的m值（moff）之比为0.54±0.04，对于GdmCl为0.57±0.1，这表明与任何一种共溶剂相互作用的位点中，超过一半埋在过渡态存在的结构集合中。
